Bottom Line:
Within this pathway, Rac is a key downstream target/effector of PI3K.On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation.Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

ABSTRACTClass I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

fig2: p85 associates with the Eps8–Abi1–Sos-1 complex under physiological conditions. (A) Top; model of the Eps8–Abi1–p85–Sos-1 complex. Bottom; lysates (10 mg) from −/− [Eps8myc] cells and supplemental materials) were immunoprecipitated (IP) with the indicated antibodies (ctr, irrelevant antibody) in the presence of the peptides PPPPPVDYTEDEE (competing) or PPPPPVAATEDEE (control). Immunoblotting (IB) was performed with the indicated antibodies. (B) Quiescent (Serum Starved) and PDGF-treated (PDGF) MEFs were fixed and stained with the indicated antibodies. Confocal analysis was carried and the apical sections are shown. Notably, colocalization between Abi1 and p85 could be detected only when the apical sections of PDGF-treated cells were analyzed. In these sections, the increase in the local concentration of the two proteins favors their detection by immunofluorescence, consistent with a notion that a relatively small (but physiologically relevant) pool of p85 is associated with Abi1 and enriched in ruffles. Arrows indicate dorsal ruffles. Bar, 10 μm.

Mentions:
To assess whether p85 is associated to the Eps8–Abi1–Sos-1 complex (Fig. 2 A), we used Eps8−/− fibroblasts in which the expression of Eps8 was restored to physiological levels (−/− [Eps8myc] cells; Scita et al., 2001; Innocenti et al., 2002). Endogenous Sos-1, Abi1, and p85 could be specifically detected in anti-myc immunoprecipitates (Fig. 2 A). In addition, the disruption of the Eps8–Abi1 interaction with the specific PPPPPVDYTEDEE peptide (but not with a control, PPPPPVAATEDEE, peptide; Mongiovi et al., 1999) caused the disappearance of Abi1, p85, and Sos-1 from the anti-myc immunoprecipitates (Fig. 2 A). Previously, we have shown that Eps8, Abi1, and Sos-1 are enriched into membrane ruffles induced by PDGF treatment (Scita et al., 2001). Similarly, endogenous p85 was found to colocalize with Abi1 (Fig. 2 B) and Eps8 (unpublished data) on treatment with PDGF. Thus, p85 is part of an Abi1-based signaling complex that includes Eps8 and Sos-1 in vivo.

fig2: p85 associates with the Eps8–Abi1–Sos-1 complex under physiological conditions. (A) Top; model of the Eps8–Abi1–p85–Sos-1 complex. Bottom; lysates (10 mg) from −/− [Eps8myc] cells and supplemental materials) were immunoprecipitated (IP) with the indicated antibodies (ctr, irrelevant antibody) in the presence of the peptides PPPPPVDYTEDEE (competing) or PPPPPVAATEDEE (control). Immunoblotting (IB) was performed with the indicated antibodies. (B) Quiescent (Serum Starved) and PDGF-treated (PDGF) MEFs were fixed and stained with the indicated antibodies. Confocal analysis was carried and the apical sections are shown. Notably, colocalization between Abi1 and p85 could be detected only when the apical sections of PDGF-treated cells were analyzed. In these sections, the increase in the local concentration of the two proteins favors their detection by immunofluorescence, consistent with a notion that a relatively small (but physiologically relevant) pool of p85 is associated with Abi1 and enriched in ruffles. Arrows indicate dorsal ruffles. Bar, 10 μm.

Mentions:
To assess whether p85 is associated to the Eps8–Abi1–Sos-1 complex (Fig. 2 A), we used Eps8−/− fibroblasts in which the expression of Eps8 was restored to physiological levels (−/− [Eps8myc] cells; Scita et al., 2001; Innocenti et al., 2002). Endogenous Sos-1, Abi1, and p85 could be specifically detected in anti-myc immunoprecipitates (Fig. 2 A). In addition, the disruption of the Eps8–Abi1 interaction with the specific PPPPPVDYTEDEE peptide (but not with a control, PPPPPVAATEDEE, peptide; Mongiovi et al., 1999) caused the disappearance of Abi1, p85, and Sos-1 from the anti-myc immunoprecipitates (Fig. 2 A). Previously, we have shown that Eps8, Abi1, and Sos-1 are enriched into membrane ruffles induced by PDGF treatment (Scita et al., 2001). Similarly, endogenous p85 was found to colocalize with Abi1 (Fig. 2 B) and Eps8 (unpublished data) on treatment with PDGF. Thus, p85 is part of an Abi1-based signaling complex that includes Eps8 and Sos-1 in vivo.

Bottom Line:
Within this pathway, Rac is a key downstream target/effector of PI3K.On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation.Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

ABSTRACTClass I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.